[unreadable] The long-term goals of this project are to contribute to the determination of the molecular mechanisms which underlie neurotransmission. Synaptic transmission is a cycle of exo- and endocytosis. During the previous project period, we established that the clathrin pathway is essential for synaptic vesicle endocytosis, even during low physiological rates of stimulation. Furthermore, we elucidated a number of the molecular interactions which are required for synaptic vesicle recycling in vivo. In order to take this work to the next level, we believe two types of approaches are required. One is to utilize biophysical methods including X-ray crystallography, surface plasmon resonance, site-directed mutagenesis and solution biochemistry to elucidate mechanisms by which these complex interactions lead to vesicle assembly and uncoating. The other is to utilize electrophysiological methods to determine the temporal order of assembly of this macromolecular complex in living synapses. Therefore the proposal is organized around the following specific aims: Aim 1: Determine the time-course of a core set of protein-protein interactions that underlie neuronal endocytosis in living synapses. Aim 2: Characterize mechanisms of clathrin coated vesicle assembly and its regulation. We will evaluate the hypothesis that the large subunits of the adaptors contain multiple clathrin binding sites, evaluate the controversial hypothesis that the FxDxF and DPF sequence motifs that are involved in direct interactions with AP-2 are also involved in direct interactions with clathrin, determine where on clathrin TD the various different types of clathrin binding sites interact, and evaluate the hypothesis that Eps15 is a ruler that sets vertex-vertex/edge-edge distances in an assembling clathrin lattice. Aim 3: Characterize mechanisms of clathrin coated vesicle uncoating. We will determine how ATP hydrolysis in the ATPase domain of Hsc70 transmits a conformational change to the substrate binding domain which causes it to either bind or release substrate, determine the nature of the interaction between auxilin and Hsc70, elucidate the mechanism by which the J-domain stimulates the ATPase activity of Hsc70, and determine the role of the 'lid' opening and closing in the Hsc70 mechanism. Successful completion of these studies will allow us to achieve a deeper level of understanding of the fundamental processes of endocytosis and neurotransmission. [unreadable] [unreadable]